During the last 15 years several laboratories have attempted to generate rabbit monoclonal antibodies, mainly because rabbits recognize antigens and epitopes that are not immunogenic in mice or rats, two species from which monoclonal antibodies are usually generated. Monoclonal antibodies from rabbits could not be generated, however, because a plasmacytoma fusion partner was not available. To obtain a rabbit plasmacytoma cell line that could be used as a fusion partner we generated transgenic rabbits carrying two transgenes, c-myc and v-abl. These rabbits developed plasmacytomas, and we obtained several plasmacytoma cell lines from which we isolated hypoxanthine/aminopterin/thymidine-sensitive clones. One of these clones, when fused with spleen cells of immunized rabbits, produced stable hybridomas that secreted antibodies specific for the immunogen. The hybridomas can be cloned and propagated in nude mice, and they can be frozen without change in their ability to secrete specific monoclonal antibodies. These rabbit-rabbit hybridomas will be useful not only for production of monoclonal antibodies but also for studies of immunoglobulin gene rearrangements and isotype switching.Monoclonal antibodies (mAbs) from rabbits have not been available because no rabbit plasmacytomas, from which a hybridoma fusion partner could be generated, have been identified. The availability of rabbit mAbs is, however, highly desirable for several reasons. First, rabbits are known to produce antibodies to many antigens that are not especially immunogenic in mice (1-5). For example, Bystryn et al. (2) directly compared rabbit and mouse antibodies directed against human melanoma cells and showed that they recognize different epitopes. Second, rabbit antibodies are generally of high affinity. Third, because most mAbs are generated in mice and rats, relatively few mAbs are available that react with mouse or rat immunogens. Because of this desire for rabbit mAbs several laboratories developed mouse-rabbit heterohybridomas, but this technology has had limited success. The earliest mouse-rabbit heterohybridomas were unstable and/or secreted only light (L) chain (6-9). Raybould and Takahashi (5) reportedly overcame this problem by using normal rabbit serum (NRS) instead of fetal calf serum (FCS) as a supplement to the culture medium. However, Verbanac et al. (10) described major problems with this method. For example, they found that the heterohybridomas were highly unstable and had to be subcloned every 4-6 weeks to avoid loss of antibody secretion. In our laboratory, we obtained no more than two to five hybridomas per fusion when using the method described by Raybould and Takahashi (5). Further, these heterohybridomas were difficult to clone, and the clones were generally unstable and did not secrete antibody over a prolonged period of time. Thus it became clear that heterohybridomas were not a satisfying solution and that rabbit-rabbit hybridomas were needed to stably produce monoclonal rabbit antibodies. We have now developed a fus...
Somatic DNA rearrangements in B lymphocytes, including V(D)J gene rearrangements and isotype switching, generally occur in cis, i. e., intrachromosomally. We showed previously, however, that 3 to 7% of IgA heavy chains have the V H and C␣ regions encoded in trans. To determine whether the trans-association of V H and C␣ occurred by trans-chromosomal recombination, by trans-splicing, or by trans-chromosomal gene conversion, we generated and analyzed eight IgA-secreting rabbit hybridomas with trans-associated V H and C␣ heavy chains. By ELISA and by nucleotide sequence analysis we found that the V H and C␣ regions were encoded by genes that were in trans in the germline. We cloned the rearranged VDJ-C␣ gene from a fosmid library of one hybridoma and found that the expressed V H and C␣ genes were juxtaposed. Moreover, the juxtaposed V H and C␣ genes originated from different IgH alleles. From the same hybridoma, we also identified a fosmid clone with the other expected product of a trans-chromosomal recombination. The recombination breakpoint occurred within the S ͞S␣ region, indicating that the trans-association of V H and C␣ genes occurred by trans-chromosomal recombination during isotype switching. We conclude that trans-chromosomal recombination occurs at an unexpectedly high frequency (7%) within the IgH locus of B lymphocytes in normal animals, which may explain the high incidence of B-cell tumors that arise from oncogene translocation into the IgH locus.Ig genes undergo somatic recombination both during early B-cell development and again during antigen-induced immune responses. The V, D, and J gene segments recombine in proB and preB cells leading to the expression of IgM and IgD on the surface of mature B cells (1). Later, during isotype switching, the VDJ genes of these B lymphocytes rearrange to downstream C H genes leading to the expression of the IgG, IgA, or IgE isotypes (2-4). Although the mechanism for class switching has not been elucidated, we know that most switch rearrangements occur in or around switch regions that are characterized by a series of tandem repeat structures found 5Ј of C , C␥, C␣, and C genes (2, 5).Although switch recombination occurs generally by intrachromosomal DNA recombination between V H and C H genes in cis, and Pernis et al. (7) identified transassociated rabbit IgG molecules in which V H and C H are derived from genes in trans. These investigators used antibodies specific for V H and C␥ allotypes and showed that, in IgH heterozygous rabbits, some of the IgG molecules had the V H allotype encoded by one IgH allele and the C␥ allotype encoded by the other IgH allele. Subsequently, Knight et al. (8) identified trans-associated secretory IgA molecules from colostrum and showed that they represented as many as 8% of total IgA molecules.The trans-association of V H and C␣ could result from transchromosomal recombination, from trans-splicing of RNA, or from trans-chromosomal gene conversion. Support for each of these mechanisms has been reported. Kipps and Herzenberg ...
Transgenic rabbits with the rabbit c-myc oncogene fused with the rabbit immunoglobulin heavy chain enhancer region (E,,) DNA were developed by microinjecting pronuclei of single cell zygotes with the gene construct and implanting the microinjected eggs into pseudopregnant females. At age 17-20 days, 3 of 21 offspring born to seven females were found to have peripheral blood leukocyte counts of >100,000 per mm3. Histology analyses showed extensive lymphocytic infiltration in the liver and kidney, indicating that these rabbits had a malignant lymphocytic leukemia. Genomic DNA analyses of thymus and peripheral blood lymphocytes revealed that the leukemic rabbits were transgenic and that both immunoglobulin heavy and K light chain genes were rearranged in the leukemic cells; thus, the leukemic cells are of B-cell lineage. One to four heavy and light chain gene rearrangements were observed, suggesting that the B-cell tumors were oligoclonal. Stable tissue culture cell lines from the bone marrow and peripheral blood of one of the transgenic rabbits have been developed. The development of B-cell leukemias in rabbits with the E,,-myc transgene contrasts with the development of B-cell lymphomas in mice carrying a similar transgene. The lymphomas in mice develop in adults and are monoclonal in origin. The leukemias in rabbits develop in juveniles, less than 3 weeks after birth, and appear oligoclonal in origin. The leukemias seem to develop in rabbit at a specific stage of development, and we suggest that a normal developmental signal may be involved in the oncogenesis.
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